Transcriptomics analysis of LINC02202/XBP1 axis in melanoma: Implications for drug targeting and PD‐1 monoclonal antibody efficacy

Abstract Malignant melanoma (MM) is a highly aggressive and deadly form of skin cancer, primarily caused by recurrence and metastasis. Therefore, it is crucial to investigate the regulatory mechanisms underlying melanoma recurrence and metastasis. Our study has identified a potential targeted regulatory relationship between LINC02202, miR‐526b‐3p and XBP1 in malignant melanoma. Through the regulation of the miR‐526b‐3p/XBP1 signalling pathway, LINC02202 may play a role in tumour progression and immune infiltration and inhibiting the expression of LINC02202 can increase the efficacy of immunotherapy for melanoma. Our findings shed light on the impact of LINC02202/XBP1 on the phenotype and function of malignant melanoma cells. Furthermore, this study provides a theoretical foundation for the development of novel immunotherapy strategies for malignant melanoma.

treatment is necessary as traditional radiotherapy and chemotherapy are not effective due to the cells' resistance, leading to a high mortality rate. 4currence and metastasis are the primary causes of death in malignant melanoma.During early metastasis, tumour cells acquire the ability to migrate, invade and spread to distant tissues and organs by crossing the surrounding stroma and entering the circulation. 5Approximately 25%-33% of melanomas arise directly from benign melanocytic nevi, which are tumours caused by the proliferation of melanocytic cells with oncogenic mutations.Melanomas occurring on exposed skin, such as the head and neck, are rarely associated with moles, indicating a fundamental genetic difference from melanomas occurring on nonexposed skin. 6The role of LINC02202 in malignant melanoma remains unknown, although it has been identified as a risk factor affecting the prognosis of hepatocellular carcinoma and potentially associated with adipogenic differentiation of ASCs. 7,8munotherapy has shown promising results in the treatment of advanced and metastatic cancers, leading to its approval as adjuvant or adjunctive therapy for non-small cell lung cancer, melanoma, triple-negative breast cancer and other solid tumours. 9Therefore, investigating the regulatory mechanisms underlying immunotherapy efficacy is of significant clinical importance.
In our study, we sequenced the long noncoding RNAs of malignant melanoma developed from melanocytic nevus at various sites and performed differential gene expression analysis.LINC02202 was identified as one of the top differentially expressed genes, and it was found to regulate the expression of PIK3R1 and FOXO1 in the PI3K signalling pathway.We also discovered a potential targeted regulatory relationship between LINC02202, miR-526b-3p and XBP1 in malignant melanoma.LINC02202 may further regulate immune infiltration and the efficacy of immune checkpoint treatments by modulating the miR-526b-3p/XBP1 signalling pathway, contributing to immune evasion, proliferation and metastasis of malignant melanoma.Our study provides insights into the effects of LINC02202/XBP1 on the phenotype and function of malignant melanoma cells, offering a theoretical basis for the development of new immunotherapy strategies for this aggressive cancer.

| Cell lines
The human foreskin fibroblasts cell line (HFF), the three human melanoma cell lines (A375, M21 and SKMEL-5) and one mouse melanoma cell lines B16 used in this study were obtained from the Chinese typical culture preservation center.These cell lines were cultured in RPMI 1640 medium (Gibco) supplemented with 10% foetal bovine serum.The cells were maintained at a temperature of 37°C in a humidified atmosphere with 5% CO 2 .

| miRNA, SiRNA and plasmid transfection
The sequences of the siRNAs and mRNA used in this study are listed in Table 1.SiRNAs targeting LINC02202 and miRNA mimics and inhibitors were designed and synthesized by Ruibo Guangzhou.The transfection methods for introducing these siR-NAs and miRNA mimics/inhibitors into cells were performed according to the references. 10Please refer to the cited reference for detailed information on the transfection methods used in this study.

Genes
Sequence TA B L E 1 The primer sequences information.

| Western blotting
After transfection, the cells were collected and lysed for further analysis.Western blot analysis was performed following the methods described in the provided references. 12The primary antibodies used in this study were diluted as follows: Cleaved-caspase-3 (1:1000, ab32042, Abcam), XBP1 (1:1500, 24,168-1-AP, Proteintech) and GAPDH (1:5000, ab9485, Abcam).Please refer to the cited references for more detailed information on the Western blot analysis methods used in this study.

| Transcriptome sequencing
Collect human tissue samples from five pairs of melanomas and three pairs of pigmented nevi, fix them with formalin, and send them to Shanghai Yuanshen Gene Company for transcriptome sequencing.Analyse based on R language pack.

| RNA extraction and quantitative real-time PCR
RNA extraction from cells or tissues was performed using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions.The extracted RNA was then subjected to quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis.The expression levels of LINC02202, has-miR-526b-3p, and RNU6 were measured using qRT-PCR with primer sets obtained from RiboBio (Guangzhou, China).The qRT-PCR method used in this study can be found in the provided reference. 13The primer sequences for qRT-PCR are listed in Table 1.

| CCK-8 proliferation assay
After transfection, cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8) assay from Promega.The CCK-8 assay measures cell viability and proliferation based on the ability of viable cells to convert a tetrazolium salt (WST-8) into a coloured formazan product.The assay was performed according to the manufacturer's instructions provided by Promega.The absorbance of the formazan product was measured using a microplate reader at the appropriate wavelength.The CCK-8 assay provides a quantitative measurement of cell proliferation and viability, allowing for the assessment of the effects of transfection on cell growth.

| Flow cytometric analyse apoptosis
Apoptosis assay was performed using the FITC Annexin V Apoptosis Detection Kit I from BD Biosciences, following the manufacturer's protocol.The assay detects apoptotic cells by labelling them with FITC-conjugated Annexin V, which binds to phosphatidylserine exposed on the outer membrane of apoptotic cells.Stained cells were then analysed using a FACSCalibur Flow Cytometer from BD Biosciences.This flow cytometer allows for the quantification and characterization of apoptotic cells based on their fluorescence properties.Please refer to the provided reference 12 for more detailed information on the apoptosis assay and flow cytometry analysis used in this study.

| Transwell assay
After processing, the cells were stained with a 0.4% crystal violet solution.This staining method allows for the visualization and quantification of cells.The invading cells were imaged using a digital microscopy system, specifically a Nikon microscope.The assay utilized 8.0 μm Transwell Permeable Supports from Corning.These Transwell inserts have a porous membrane with a pore size of 8.0 μm, allowing for the study of cell invasion through the membrane.The stained cells were observed and imaged using the digital microscopy system, enabling the analysis of cell invasion.
After 48 h of incubation, the cells were subjected to a luciferase reporter assay.This assay allows for the quantification of luciferase activity as an indicator of gene regulation.Please refer to the provided reference 13 for more detailed information on the luciferase reporter assay used in this study.

| Transplanted tumour model
In the transplanted tumour model, B16 cells that were stably transfected with lentiviruses were constructed in the laboratory.These transfected B16 cells were cultured and prepared for injection.C57 mice were divided into four groups, with five mice in each group.
Each mouse was injected with 5 × 10 6 cells in a volume of 100 μL.
Additionally, the mice received anti-PD1 antibody treatment at a dose of 200 μg per mouse.The experiment was conducted until the tumour volume reached 1500 mm 3 , at which point the mice were sacrificed 15 days after injection. 12All animal experiments were performed in accordance with the ethical policies and procedures approved by the Laboratory Animal Welfare and Ethics Committee of the Ningxia medical university.

| Statistical analysis
For statistical analyses, unpaired Student's t-tests or one-way ANOVA followed by Duncan's multiple range tests were performed using Prism 8.0 software.A p-value less than 0.05 was considered statistically significant.Kaplan-Meier survival analysis was used to calculate the overall survival rate of melanoma.This analysis helps assess the impact of different treatments on the survival of mice in the study.

| LINC02202 is highly expressed in melanoma patients' tissues
Based on the sequencing results from three melanocytic nevi tissues and five melanoma patients' tissues, bioinformatics analysis was performed.A heat map was generated to visualize the differential expression of all noncoding RNAs between the two groups (Figure 1A).PCA analysis was conducted to demonstrate the distinction between melanoma and melanocytic nevi (Figure 1B).Volcano plot analysis and lncRNA differential analysis identified LINC02202 as a highly expressed lncRNA in melanoma patients compared to the melanocytic nevi group (Figure 1C).LINC02202 ranked among the top 10 highly expressed lncRNAs, with a statistically significant p-value of 0.000781 (Figure 1D).

| LINC02202 suppress melanoma cell growth and metastasis
The relative expression of LINC02202 was found to be significantly tumourigenesis, the A375 and SKMEL-5 cell lines, which exhibited intermediate expression of LINC02202, was used.SiRNA and LINC02202 were transfected into A375 and SKMEL-5 cells, and LINC02202 expression was detected 72 h post transfection (Figure 2B).CCK-8 assays revealed that cell viability was significantly reduced in LINC02202overexpressing cells (Figure 2C).Migration assays showed a higher migration rate in LINC02202-overexpressing cells, while a lower migration rate was observed when LINC02202 was knocked down (Figure 2D).
Western blotting further supported these findings, as the expression of Cleaved-caspase-3 was induced in the siLINC02202 group and suppressed in the LINC02202-overexpressing group (Figure 2E).

| MiR-526b-3p is an important target of LINC02202
In our study, we utilized the TCGA database to search for miRNA expression in melanoma samples.A heatmap was generated to visualize the miRNA expression in each sample (Figure 3A).Correlation analysis was performed between each miRNA and LINC02202, resulting in the identification of two miRNAs: hsa-miR-2681 and has-miR-526b-3p (Figure 3B-D).While hsa-miR-2681 has limited research, it is almost nonexpressed in the TCGA database, so we focused on has-miR-526b-3p, which has been reported in glioma and colorectal cancer but rarely in melanoma.We transfected LINC02202 or siLINC02202 into SKMEL-5 cells and detected the expression level of miR-526b-3p 72 h post transfection, finding that miR-526b-3p expression increased when LINC02202 was disturbed (Figure 3E).A luciferase reporter assay demonstrated significantly lower luciferase activity in the miR-526b-3p mimic+ LINC02202-wt group compared to the miR-526b-3p mimic+ LINC02202-mut group (Figure 3F).Additionally, there was greater LINC02202 enrichment in the bio-miR-526b-3p group, indicating a strong correlation (Figure 3F).Furthermore, survival analysis based on TCGA data showed a positive correlation between miR-526b-3p expression and good prognosis (Figure 3G).

| MiR-526b-3p could target XBP1
In our study, we conducted an analysis of mRNA expression differences in first-related genes by analysing our sequencing results.A heatmap was generated to provide an overview of the differentially expressed genes (Figure 5A).PCA analysis was performed to visualize the differences in mRNA expression between melanoma and melanocytic nevi (Figure 5B).Additionally, a volcano plot analysis was conducted to identify differentially expressed mRNAs in melanoma patients compared to the melanocytic nevi group (Figure 5C).
By aligning the sequencing data with TARGETSCAN analysis, we identified three genes of interest: XBP1, c8orf31 and c10orf25 (Figure 5D).Survival analysis based on TCGA data demonstrated that XBP1 was positively correlated with poor prognosis (Figure 5E, p = 0.013).However, there was no observed correlation between the expression of c8orf31 and c10orf25 and survival.Consequently, we further investigated the regulatory mechanism of XBP1 as a target gene.

| LINC02202 promotes melanoma progression by miR-526b-3p/XBP1 axis
To investigate the regulatory relationship between miR-526b-3p and XBP1, we conducted a luciferase reporter assay that confirmed the direct targeting of the 3'UTR of XBP1 by miR-526b-3p.This was demonstrated by significantly lower luciferase activity in the miR-526b-3p mimics+XBP1-wt group (Figure 6A).These findings indicate that miR-526b-3p inhibits XBP1 expression by directly targeting its 3'UTR.We also examined the relationship between LINC02202 and XBP1.Our results showed that not only miR-526b-3p can affect the expression of XBP1, but LINC02202 also influences the expression of XBP1.Moreover, the upregulation of miR-526b-3p can block the expression of XBP1 induced by LINC02202 at both the mRNA (Figure 6B) and protein (Figure 6C) levels.Furthermore, we observed a positive correlation between the expression of LINC02202 and XBP1 (Figure 6D).Finally, we tested the correlation between XBP1 and PD-L1 expression using 30 human samples (patient information in Table 2) and found a positive correlation between XBP1 and PD-L1 expression (Figure 6E).In summary, LINC02202 can regulate XBP1 expression by targeting miR-526b-3p.

| Inhibiting LINC02202/XBP1 can increase the efficacy of PD-1 immunotherapy in melanoma
In our animal experiments, we observed that tumour volume and tumour weight were significantly decreased when shLINC02202/ shXBP1 were combined with anti-PD-1 treatment (Figure 7A,B).
On the contrary, overexpression of LINC02202 promoted tumour growth.Furthermore, inhibiting XBP1 weakened the expression of Ki-67, which was induced by LINC02202 (Figure 7C).Additionally,

| DISCUSS ION
Indeed, long noncoding RNAs (lncRNAs) have been recognized as important regulators of various cellular processes, including those involved in cancer development and progression, such as melanoma.
[15][16] The tissue-and disease-specific expression patterns of ln-cRNAs make them potential specific markers for non-invasive diagnosis of diseases, including cancer. 179][20] Melanoma, being a well-studied and genetically defined tumour, has been  microenvironment play crucial roles in controlling the growth of moles and preventing their progression to melanoma. 21,22Studies have shown that the rate of progression from mole to melanoma may be slightly higher in transplant recipients, suggesting the involvement of the immune system. 23Histological evidence indicates that melanomas exhibit stronger lymphocytic infiltration and cytotoxic responses compared to pigmented nevi. 22,232][23][24] Further investigation is required to elucidate these interactions and mechanisms.
Research has found that IRE1 α/KIRA8, an inhibitor of the XBP1 signalling pathway, can significantly inhibit tumour growth and has a better therapeutic effect when combined with PD-1 antibodies. 25Immunotherapy has shown promising results in the treatment of melanoma, but many mechanisms underlying its efficacy are still unclear.In our study, we found that LINC02202 is highly expressed in melanoma and can regulate the expression of miR-526b-3p/XBP1/PD-L1 axis.In vitro experiments revealed that disturbance of LINC02202 inhibited the viability, proliferation and metastasis of melanoma cells (SKMEL-5 and A375).
Animal experiments further confirmed that LINC02202 may regulate the efficacy of immunotherapy in melanoma through the XBP1 pathway.
In summary, our findings suggest that LINC02202 plays a role in activating melanoma cell viability and metastasis while higher in the three melanoma cell lines (A375, M21 and SKMEL-5) compared to the normal cell line human foreskin fibroblasts (HFF) (Figure 2A).To investigate the function of LINC02202 in melanoma F I G U R E 1 LINC02202 is increased in melanoma patients.(A) Heat map showing melanocytic nevi tissue and five melanoma patients' tissues groups all noncoding RNA expression.(B) PCA analysis showed the difference between the melanoma and melanocytic nevi.(C) Volcano plot analysis and lncRNA differential analysis of the differentially expressed noncoding RNAs.(D) The top seven list of increased and decreased expression difference analysis.
findings collectively indicate that overexpression of miR-526b-3p can suppress melanoma progression.F I G U R E 2 LINC02202 suppress melanoma cell viability, apoptosis and metastasis.(A) The RT-PCR detected the relative expression of LINC02202 in the three melanoma cell lines (A375, M21 and SKMEL-5) and the normal cell line human foreskin fibroblasts (HFF).(B) We transfected siRNA and LINC02202 in A375 and SKMEL-5 cells and using RT-PCR detected LINC02202 expression at 72 h post transfection.(C) CCK-8 assays analysed the effect of LINC02202 on cell viability.(D) Transwell assays analysed the effect of LINC02202 on cell migration.(E) The Western blotting assay analysed the effect of LINC02202 on cell apoptosis.*p < 0.05, **p<0.01,***p < 0.001.F I G U R E 4 MiR-526b-3p could regulate melanoma progression.(A, B) The RT-PCR detected miR-526b-3p expression levels 72 h post transfection.(C) CCK-8 assays analysed the effect of miR-526b-3p on cell viability.(D) Transwell assays analysed the effect of miR-526b-3p on cell migration.(E) The Flow Cytometry assay analysed the effect of LINC02202 on cell apoptosis.*p < 0.05, **p<0.01.F I G U R E 3 LINC02202 can target MiR-526b-3p.(A) The miRNA expression of each sample in the TCGA database were shown by hot map.(B) The correlation analysis between each miRNA and LINC02202.(C) The intersection of correlation analysis, NCBI Blast results and TCGA database prediction analysis results.(D) Predicted binding site for 3'UTR of hsa-miR-2681 and has-miR-526b-3p and LINC02202 sequences.(E) The RT-PCR detected the miR-526b-3p expression level 72 h post transfection.(F) Luciferase reporter assay for LINC02202 that directly targets miR-526b-3p.(G) The impact of miR-526b-3p on the survival and prognosis of melanoma by TCGA database.*p < 0.05, **p<0.01.

F I G U R E 5
MiR-526b-3p could target XBP1.(A) The heatmap shows an overview of differentially expressed genes.(B) PCA analysis the difference of mRNA between the melanoma and melanocytic nevi.(C) Volcano plot analysis differentially expressed mRNAs in melanoma patients and the melanocytic nevi group.(D) Venn analysis of sequence data with targetscan.(E) The survival analysis the correction of XBP1 prognosis by Kaplan-Meier, data obtained from TCGA.(F) The survival analysis the correction of C8orf31 and C10orf25 prognosis by Kaplan-Meier, data obtained from TCGA.shXBP1 enhanced the infiltrate of CD8+ T cells (Figure7D).The expression of LINC02202 and XBP1 showed in FigureS1.Overall, our findings suggest that LINC02202 can promote melanoma progression and inhibit T cell immune killing of melanoma cells through the miR-526b-3p/XBP1 pathway.
used as a model to investigate the growth arrest of lesions following oncogene mutations.The immune system and the tumour F I G U R E 6 LINC02202 could regulate miR-526b-3p/XBP1 axis.(A) The luciferase reporter assay for miR-526b-3p that directly targets the 3'UTR of XBP1.(B) The RT-PCR detected that LINC02202 could regulate miR-526b-3p/XBP1 axis.(C) The Western blotting detected that LINC02202 could regulate miR-526b-3p/XBP1 axis.(D) Correlation analysis between LINC02202 and XBP1 expression.(E) Immunohistochemical detection of XBP1 and PD-L1 expression in human melanoma tissue.**p<0.01.TA B L E 2 General characteristics of malignant melanoma patients.